Biocide composition

ABSTRACT

Biocide compositions comprising 5-chloro-2-methyl-4-isothiazolin-3-one-and 2-methyl-4-isothiazolon-3-one- in a weight ratio 1.2 to 25.4 : 74.6 to 98.8 demonstrate synergism and decreased sensitization potential.

This invention relates to biocide compositions of5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one(also known as 5-chloro-2-methyl-3-isothiazolone and2-methyl-3-isothiazolone, respectively) which at specific use levelsexhibit not only synergy but also exhibit a much lower incidence ofsensitization

The search for ways to lower sensitization to biocides is a continuingone. All or nearly all commercially available preservatives possess skinsensitization potential.

The biocides and their preparations are disclosed in U.S. Pat. Nos.3,761,488; 4,150,026; 4,396,413; 4,505,889 and 3,769,315.

Kathon biocide (886) a widely used, broad spectrum antimicrobial agent,contains, as active ingredients (a.i.), a mixture of5-chloro-2-methyl-4-isothiazolin-3-one ("651") and2-methyl-4-isothiazolin-3-one ("573") in an approximate ratio of 3:1,respectively, with magnesium chloride (9%) and magnesium nitrate (16%)present as stabilizers. The biocide is an effective preservative fortoiletry formulations, cosmetic and household cleaning products. It isalso used in some heavy industrial applications such as cooling-towerwater, metalworking fluid, and latex emulsions.

A variety of industrial chemicals, cosmetic fragrances, and therapeuticagents, as well as several naturally occurring substances such as poisonivy, can cause delayed contact dermatitis in animals and humans. Thesesensitizers fall into diversified chemical categories ranging fromsimple inorganic metals, such as nickel, to complex organic chemicals.

Several articles on the description of how tests are conducted and whichrelate to isothiazolones are: "Demonstration of Kathon® CG in somecommercial products", Contact Dermatitis 1986: 15: 24-27; "Allergiccontact dermatitis due to a biocide containing5-chloro-2-methyl-4-isothiazolin-3-one", Contact Dermatitis 1986: 14:201-204; "Patch test sensitivity to Kathon® CG", Contact Dermatitis1986: 14: 155-157; "Contact allergy to the preservative Kathon ® CG",Contact Dermatitis 1986: 14: 85-90; "Immediate and delayed reactions tocosmetic ingredients", Contact Dermatitis 1985: 13: 258-265; "Diagnosticpatch test concentration for Kathon CG", Contact Dermatitis 1985: 13:242-245; "Biocide patch tests", Contact Dermatitis 1985: 12: 99-103; and"Patch tests with fragrance materials and preservatives", ContactDermatitis 1985: 12: 87-92.

In the effort to find a way to decrease sensitization while keeping theeffectiveness at an acceptable level it was found that it is possible touse specific combinations of 651 and 573 which decrease thesensitization by a factor of about 15 to 20 while decreasing theactivity by a factory of only about 3. It was further discovered thatthe composition demonstrated unexpected activity between various userates.

The invention relates specifically to a microbicidal composition, whichhas the same uses as the present commercial isothiazolones, comprising asynergistic mixture the first component of which is5-chloro-2-methyl-4-isothiazolin-3-one (651) and the second component ofwhich is 2-methyl-4-isothiazolin-3-one (573). The synergisticcompositions have from about 1.2 to about 25.4% of5-chloro-2-methyl-4-isothiazolin-3-one (651) with the amount of fromabout 2.3 to about 8.5% of 651 being preferred and an amount of fromabout 2.3 to about 4.5% of 651 being most preferred.

The invention also relates to a method of inhibiting the growth ofbacteria, fungi or algae in a locus subject to contamination bybacteria, fungi or algae, which comprises incorporating into or onto thelocus in an amount which is effective to adversely affect the growth ofbacteria, fungi or algae the synergistic combination of 651 and 573.

The composition of the invention can be formulated as solutions inwater. While the amount of the instant composition in the formulatedsolution can vary over a wide range, the solutions can conveniently beformulated to contain from about 20 to about 100 ppm of the compositionin solution, with the preferred range being from about 25 to 50 ppm ofthe composition. In formulating the solutions, other solvents which arewater-miscible, such as ethyl alcohol, propyl alcohol, isopropylalcohol, diethylene glycol, dipropylene glycol, polyethylene glycol,diethylene glycol ethyl ether, and the like, may be employed in order toaid in solubizing the active components. Furthermore, various otherconventional additives may be employed, such as surfactants, dispersingagents, corrosion inhibitors, and the like.

Potential areas of general application of these synergistic combinationsinclude disinfectants, sanitizers, cleaners, deodorizers, liquid andpowder soaps, skin removers, oil and grease removers, food processingchemicals, dairy chemicals, food preservatives, animal foodpreservatives, wood preservation, aqueous dispersions of organicpolymers, paint, lazures, stains, mildewcides, hospital and medicalantiseptics, metal working fluids, cooling water, air washers, petroleumproduction, paper treatment, paper mill slimicides, petroleum products,adhesives, textiles, pigment slurries, latexes, leather and hidetreatment, petroleum fuel, heating oil, laundry sanitizers, agriculturalformulations, inks, mining, nonwoven fabrics, petroleum storage, rubber,sugar processing, tobacco, swimming pools, cosmetics, toiletries,pharmaceuticals, chemical toilets, household laundry products, dieselfuel additives, waxes and polishes and many other applications wherewater and organic materials come in contact under conditions which allowthe growth of undesired microorganisms.

Other known biocides may be combined advantageously with theisothiazolones of this invention to afford a biocide having broaderactivity.

The products of this invention are especially useful as preservativesfor products which may be in contact with the skin such as cosmetics,toiletries and the like.

The synergistic 573/651 combinations provide long-lasting activity forcontrolling a wide spectrum of microorganisms at a small fraction ofconcentrations needed by either 573 or 651 alone.

PREPARATION OF COMBINATIONS

The following examples illustrate the preparation of the combinations.They can be prepared by starting with substantially pure 651 and 573 orby adding substantially pure 573 to 886.

EXAMPLE 1

To 179 g monochlorobenzene (MCB) were co-fed chlorine and a 25% solutionof N-methyl-3-mercaptopropionamide (MMPA) in MCB at a molar ratio ofabout 1.4 in about 105 minutes at 50° C. with a good mixing till 1.5mole MMPA was fed. At the end of co-feed, the chlorine feed wascontinued to a final molar feed ratio of 2.0.

The chlorination slurry was filtered, washed with an appropriate solventand dried to afford the hydrochloride salt of:

    ______________________________________                                                   573 =  61.3%                                                                  651 =  2.74%                                                                  Ratio =                                                                              95.7:4.3                                                    ______________________________________                                    

The above hydrochloride product was slurried in water and wasneutralized to pH 4 by adding gradually solid sodium carbonate. Thesolution was filtered to obtain the product containing:

    ______________________________________                                                   573 =  28.8%                                                                  651 =  1.2%                                                                   Ratio =                                                                              96:4                                                        ______________________________________                                    

EXAMPLE 2

To a stirred 1.0 mole solution of 25% MMPA in MCB was passed 1 moleanhydrous hydrogen chloride at 45° C. in 1 hour. Next the mixture waschlorinated by adding 2 moles of chlorine over a period of 2.5 hourswhile agitating and maintaining the batch temperature around 45° C. Atthe end of chlorination, the mixture was degassed for 20 minutes toremove free hydrogen chloride. The chlorination slurry was thenfiltered, washed with MCB and dried to afford the hydrochloride salt of:

    ______________________________________                                                   573 = 60.4%                                                                   651 = 3.0%                                                         ______________________________________                                    

The above hydrochloride salt was slurried in water and neutralized to pH6.5 by gradually adding sodium bicarbonate powder at 20° C. to afford anaqueous solution of product:

    ______________________________________                                                   573 =  29.8%                                                                  651 =  1.5%                                                                   Ratio =                                                                              95.2:4.8                                                    ______________________________________                                    

EXAMPLE 3

By employing the method used in Example 1, but by substituting MMPA withan equimolar quantity of pure N,N'-Dimethyldithiodipropionamide andco-feeding chlorine at a molar ratio of 2.25, and continuing chlorinefeed to a final molar feed ratio of 3.0, afforded the hydrochlorideproduct:

    ______________________________________                                                   573 =  60.3%                                                                  651 =  3.2%                                                                   Ratio =                                                                              95:5                                                        ______________________________________                                    

The hydrochloride product was neutralized as in Example 1 above toafford an aqueous solution with the 573:651 ratio of 95:5.

EXAMPLE 4

By employing the method used in Example 2 above, but substituting MMPAwith an equimolar quantity of pure N,N'-dimethyldithiodipropionamide andadding 3 equivalents of chlorine instead of 2 equivalents, there isobtained the hydrochloride salt, which was filtered, washed andreslurried in water. The aqueous slurry was neutralized to afford theaqueous product with the following composition:

    ______________________________________                                                   573 =  30.5%                                                                  651 =  0.9%                                                                   Ratio =                                                                              97:3                                                        ______________________________________                                    

EXAMPLE 5

The filtered and washed chlorination slurry from Example 4 wasreslurried in an appropriate solvent and neutralized with anhydrousammonia. The ammonium chloride was filtered off and washed with thesolvent. The filtrate and washing were combined and stripped (35°/10 mmHg) to afford the combination containing 92.1% of 573 and 4.8% of 651.(Ratio=95:5.)

EXAMPLE 6

By dissolving 1.45 g of substantially pure 573 (see example 7) and 0.045g of 886 in 98 g water a combination of the following composition isprepared:

    ______________________________________                                                   573 =  1.466%                                                                 651 =  0.055%                                                                 Ratio =                                                                              96.4:3.6                                                    ______________________________________                                    

EXAMPLE 7 Preparation of substantially pure 573 solution

The hydrochloride obtained in Example 1 was dissolved in 500 g ofboiling methanol and gradually cooled to 10° C. The crystal slurry wasfiltered and washed with cool solvent and dried in vacuum to afford132.8 g of 573 hydrochloride containing 74.9% free base and 24.1% HCl(99.0%) with no 651.

45 g of the above hydrochloride salt was dissolved in 135 g water andneutralized to pH 4.2 by adding a 20% aqueous sodium carbonate solutionto afford an aqueous solution containing:

573=12.5%

651=<0.1% 4,5dichloro-2-methyl-4-isothiazolin-3-one=<0.1

EXAMPLE 8 Preparation of substantially pure 573

The slurry of 354 g (1.5 moles) of N,N'-dimethyldithiodipropionamide in2 1. butyl acetate was cooled to -20° C., and under its surface waspassed 335 g (4.72 moles, 3.14 equiv.) of chlorine over a period of 100minutes. The mixture was allowed to warm to 15° C. when an exchangereaction occurred. The batch was held below 50° C. and allowed to standat room temperature for 1 hour. Next, the mixture was filtered and thewet cake washed with 200 ml butyl acetate and dried in vacuum. The 472 gcrude hydrochloride salt was recrystallized from methanol to affordsubstantially pure 573 hydrochloride salt (99%).

This was dissolved in water and neutralized with potassium carbonate toa pH 7.5 to afford a 25.0% product solution.

The above solution was extracted with ethylene dichloride (EDC, 1×800ml, 2×300 ml). The EDC extracts were combined and the solvent removed toafford 241 g of 99.7% pure 573.

EXAMPLE 9 Preparation of substantially pure 651

A chlorination slurry obtained by the chlorination ofN-methyl-3-mercaptopropionamide in toluene by co-feeding it withchlorine at a molar feed ratio of 3.0 was filtered, washed and dried toafford a hydrochloride salt containing:

    ______________________________________                                                  573 =  19.8%                                                                  651 =  57.8%                                                                  Ratio =                                                                              25.5:74.5                                                    ______________________________________                                    

The hydrochloride salt, (225 g), was slurried in 870 ml of ethyl etherand to it was added dropwise 54.3 g of pyridine (0.686 moles, the amountrequired to neutralized 80% of 651 hydrochloride). The mixture wasfiltered and the solids washed with ethyl ether. The filtrate andwashings were combined and stripped (35°/20mm Hg) to afford 90 g of paleyellow crystalline solids containing 98.5% 651.

EVALUATION OF SYNERGY

The synergism of the combinations is demonstrated by testing at a widerange of concentrations and ratios of the two components generated by2-fold serial dilutions in a growth medium (Trypticase Soy Broth or TSB,Difco) of 573 in one dimension and 886 in a second dimension, against amixture of the following 12 microorganisms: Escherichia coli (ATCC11229) Candida albicans (ATCC 11651), Pseudomonas oleovarans (ATCC8062), Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC6538), Enterobacter aerogenes (ATCC 15038), Pseudomonas cepacia (ATCC17765), Pseudomonas putida (ATCC 795), Aspergillus niger (ATCC 6275),Talaromyces luteus (ATCC 10465), Penicillum oxalicum (ATCC 24784) andSerratia marcescens (a shampoo isolate). Each single culture wasinoculated to TSB agar slants and incubated for 2 days at 30° C. for C.albicans and all bacteria except E. coli and S. aureus, which were at37° C., and incubated for 10-14 days at 25° C. for fungal cultures. Thebacteria and fungi were then washed off the slants with phosphate buffer(0.125 M, pH 7.2) and inoculated into each test tube containing a givenconcentration and ratio of 573 and 886 to make final cell concentrationof about 2×10⁷ bacteria and 2×10⁶ fungi per ml. The lowest concentrationof the two components, acting alone or in a mixture, to visibly inhibitcell growth at 30° C. for over 7 days is the minimum inhibitoryconcentration (MIC). Seven days after inoculation, 5 microliter ofsample from each test tube (containing about 10⁵ bacteria and 10⁴ fungifrom original inoculation) was removed and assessed for viable organismsby 1:50 dilution into TSB medium and checked for regrowth. No viableorganisms were detected from the test tubes where MICs were observedThus, the MICs showed very good control of those organisms and weretaken as end points of activity. Each test tube was reinoculated 2 moretimes (a total of 3 inoculations) to determine the capacity and thelong-lasting activity of the combinations.

                                      TABLE 1                                     __________________________________________________________________________    Treatments (ppm)                                                                            Experiment No. 1                                                                          Experiment No. 2                                             Ratio                                                                              Growth after inoculation                                                                  Growth after inoculation                            573 (A)                                                                            886 (B)                                                                           A/B  1st 2nd 3rd 1st 2nd 3rd                                         __________________________________________________________________________    500.0                                                                              0.0 0    -   -   -   -   -   -                                           250.0                                                                              0.0 0    -   +   +   -   -   -                                           125.0                                                                              0.0 0    +   +   +   -   -   -                                           62.5 0.0 0    +   +   +   +   +   +                                           31.2 0.0 0    +   +   +   +   +   +                                           15.6 0.0 0    +   +   +   +   +   +                                           9.8  0.0 0    +   +   +   +   +   +                                           3.9  0.0 0    +   +   +   +   +   +                                           2.0  0.0 0    +   +   +   +   +   +                                           1.0  0.0 0    +   +   +   +   +   +                                           0.5  0.0 0    +   +   +   +   +   +                                           250.0                                                                              0.3 1000 -   +   +   -   -   -                                           250.0                                                                              0.5 500  -   -   +   -   -   -                                           125.0                                                                              0.3 500  +   +   +   -   -   -                                           250.0                                                                              1.0 250  -   -   -   -   -   -                                           125.0                                                                              0.5 250  -   +   +   -   -   -                                           62.5 0.3 250  +   +   +   +   +   +                                           250.0                                                                              2.0 125  -   -   -   -   -   -                                           125.0                                                                              1.0 125  -   +   +   -   -   -                                           62.5 0.5 125  +   +   +   +   +   +                                           31.2 0.3 125  +   +   +   +   +   +                                           250.0                                                                              4.0 62.5 -   -   -   -   -   -                                           125.0                                                                              2.0 62.5 -   -   -   -   -   -                                           62.5 1.0 62.5 +   +   +   -   -   -                                           71.2 0.5 62.5 +   +   +   +   +   +                                           15.6 0.3 62.5 +   +   +   +   +   +                                           250.0                                                                              8.0 31.2 -   -   -   -   -   -                                           125.0                                                                              8.0 31.2 -   -   -   -   -   -                                           62.5 2.0 31.2 -   -   -   -   -   -                                           31.2 1.0 31.2 +   +   +   -   -   -                                           15.6 0.5 31.2 +   +   +   +   +   +                                           7.8  0.3 31.2 +   +   +   +   +   +                                           250.0                                                                              16.0                                                                              15.6 -   -   -   -   -   -                                           125.0                                                                              8.0 15.6 -   -   -   -   -   -                                           62.5 4.0 15.6 -   -   -   -   -   -                                           31.2 2.0 15.6 -   -   -   -   -   -                                           15.6 1.0 15.6 +   +   +   +   +   +                                           7.8  1.5 15.6 +   +   +   +   +   +                                           3.9  0.3 15.6 +   +   +   +   +   +                                           125.0                                                                              16.0                                                                              8    -   -   -   -   -   -                                           62.5 8.0 8    -   -   -   -   -   -                                           31.2 4.0 8    -   -   -   -   -   -                                           15.6 2.0 8    -   +   +   -   +   +                                           7.8  1.0 8    +   +   +   +   +   +                                           3.9  0.5 8    +   +   +   +   +   +                                           2.0  0.3 8    +   +   +   +   +   +                                           62.5 16.0                                                                              4    -   -   -   -   -   -                                           31.2 8.0 4    -   -   -   -   -   -                                           15.6 4.0 4    -   +   +   -   +   +                                           7.8  2.0 4    -   +   +   +   +   +                                           3.9  1.0 4    +   +   +   +   +   +                                           2.0  0.5 4    +   +   +   +   +   +                                           1.0  0.3 4    +   +   +   +   +   +                                           31.2 16.0                                                                              2    -   -   -   -   -   -                                           15.6 8.0 2    -   -   +   -   +   +                                           7.8  6.0 2    -   +   +   -   +   +                                           3.9  2.0 2    +   +   +   +   +   +                                           2.0  1.0 2    +   +   +   +   +   +                                           1.0  0.5 2    +   +   +   +   +   +                                           0.5  0.3 2    +   +   +   +   +   +                                           15.6 16.0                                                                              1    -   -   -   -   -   +                                           7.8  8.0 1    -   +   +   -   +   +                                           3.9  6.0 1    -   +   +   +   +   +                                           2.0  2.0 1    +   +   +   +   +   +                                           1.0  1.0 1    +   +   +   +   +   +                                           0.5  0.5 1    +   +   +   +   +   +                                           7.8  16.0                                                                              0.5  -   +   +   -   +   +                                           3.9  8.0 0.5  -   +   +   +   +   +                                           2.0  4.0 0.5  +   +   +   +   +   +                                           1.0  2.0 0.5  +   +   +   +   +   +                                           0.5  1.0 0.5  +   +   +   +   +   +                                           3.9  16.0                                                                              0.25 -   +   +   -   +   +                                           2.0  8.0 0.25 -   +   +   +   +   +                                           1.0  4.0 0.25 +   +   +   +   +   +                                           0.5  2.0 0.25 +   +   +   +   +   +                                           2.0  16.0                                                                              0.13 -   +   +   -   +   +                                           1.0  8.0 0.13 +   +   +   +   +   +                                           0.5  4.0 0.13 +   +   +   +   +   +                                           1.0  16.0                                                                              0.06 -   +   +   -   +   +                                           0.5  8.0 0.06 +   +   +   +   +   +                                           0.5  16.0                                                                              0.03 -   +   +   -   +   +                                           0.0  32.0                                                                              0    -   -   -   -   -   -                                           0.0  16.0                                                                              0    -   +   +   -   +   +                                           0.0  8.0 0    +   +   +   +   +   +                                           0.0  4.0 0    +   +   +   +   +   +                                           0.0  2.0 0    +   +   +   +   +   +                                           0.0  1.0 0    +   +   +   +   +   +                                           0.0  0.5 0    +   +   +   +   +   +                                           0.0  0.3 0    +   +   +   +   +   +                                           0.0  0.0 0    +   +   +   +   +   +                                           __________________________________________________________________________     - growth inhibited                                                            + growth not inhibited                                                   

Synergism is determined by the industrially accepted method described byKull, F.C.; Eisman, P.C.; Sylvestrowicz, H.D. and Mayer, R.L. in AppliedMicrobiology 9:538-541 (1961) using the ratio determined by ##EQU1##wherein

QA=concentration of compound A in parts per million (ppm), acting alone,which produced an end point

Qa=concentration of compound A in ppm, in the mixture, which produced anend point

QB=concentration of compound B in ppm, acting alone, which produced anend point

Qb=concentration of compound B in ppm, in the mixture, which produced anend point

When the sum of Qa/QA plus Qb/QB is greater than one, antagonism isindicated. When the sum is equal to one, additivity is indicated, andwhen the sum is less than one, synergism is indicated.

The test results for demonstration of synergism between 573 and 651 areshown in Table 1. As shown, a wide range of concentration and ratios wastested. More importantly all the critical end-point activities for thecombinations were covered. The combinations that consistently providecontrol for all three inoculations are considered most effective. Thus,the end-point activities are based on the minimum concentrations of agiven ratio of combination product that inhibit all three inoculations.

Table 2 shows the calculation of synergism between 573 and 651combinations. The end-point activity (MIC in ppm) is taken from Table 1.

QA is the end-point activity of 573 alone

QB is the end-point activity of 886 alone

Qa is the end-point activity 573 in the mixture of 573 and 886

Qb is the end-point activity of 886 in the mixture of 573 and 886.

Since 886 is 25% 573 and 75% 651, the total 573 (Qa') in thecombinations is Qa +25% Qb, and the total 651 (Qb') in the combinationsis 75% Qb. Total concentration of 573 and 651 in the mixture is Qa' andQb' The % 573 in the mixture is Qa'/(Qa'+Qb'), and %651 in the mixtureis Qb'/(Qa'+Qb'.)

Calculation of the synergy index (SI) for 573 and 651 combinations isbased on data in Table 1 that shows 573, at very low concentrations (<8ppm), does not contribute significantly to the overall activity of 886.In other words, the activity of 886 at most recommended use rates (<30ppm) is essentially the activity of 651. Therefore, the calculation ofsynergy index (SI) is Qa'/QA +Qb'/0.75 QB. The most preferred synergyfor 573 and 651 combinations is seen at 2.3%-4.5% of 651. Taking thesynergy index of less than 0.5 as most significant, then a range of 2.3%to 8.5% of 651 in the 573/651 combinations is preferred.

The following Table 2 shows the end-points and the results of thecalculations.

                                      TABLE 2                                     __________________________________________________________________________    Calculation of Synergism of 573 and 651 Combinations                                           Calculations                                                 End-point        Total                                                                             Total                                                                             Total                                                Expt.                                                                             Activity (MIC) in ppm                                                                      573 651 ppm   %   %                                          No. QA QB Qa  Qb Qa' Qb' Qa' + Qb'                                                                           573 651                                                                              Qa'/QA                                                                             Qb'/QB*                                                                            SI                            __________________________________________________________________________    1   500                                                                              32 500.0                                                                             0  500.0                                                                             0.0 500.0 100.0                                                                             0.0                                                                              1.00 0.00 1.00                              500                                                                              32 250.0                                                                             1  250.2                                                                             0.7 251.0 99.7                                                                              0.3                                                                              0.50 0.03 0.53                              500                                                                              32 250.0                                                                             2  250.5                                                                             1.5 252.0 99.4                                                                              0.6                                                                              0.50 0.06 0.56                              500                                                                              32 125.0                                                                             2  125.5                                                                             1.5 127.0 98.8                                                                              1.2                                                                              0.25 0.06 0.31                              500                                                                              32 62.5                                                                              2  63.0                                                                              1.5 64.5  97.7                                                                              2.3                                                                              0.13 0.06 0.19                              500                                                                              32 31.2                                                                              2  31.7                                                                              1.5 33.2  95.5                                                                              4.5                                                                              0.06 0.06 0.13                              500                                                                              32 31.2                                                                              4  32.2                                                                              3.0 35.2  91.5                                                                              8.5                                                                              0.06 0.13 0.19                              500                                                                              32 31.2                                                                              8  33.2                                                                              6.0 39.2  84.7                                                                              15.3                                                                             0.07 0.25 0.32                              500                                                                              32 31.2                                                                              16 35.2                                                                              12.0                                                                              47.2  74.6                                                                              25.4                                                                             0.07 0.37 0.44                              500                                                                              32 15.6                                                                              16 19.6                                                                              12.0                                                                              31.6  62.1                                                                              37.9                                                                             0.04 0.50 0.54                              500                                                                              32 0.0 32 8.0 24.0                                                                              32.0  25.0                                                                              75.0                                                                             0.02 1.00 1.02                          2   125                                                                              32 125.0                                                                             0  125.0                                                                             0.0 125.0 100.0                                                                             0.0                                                                              1.00 0.00 1.00                              125                                                                              32 62.5                                                                              1  62.7                                                                              0.7 63.5  98.8                                                                              1.2                                                                              0.50 0.03 0.53                              125                                                                              32 31.2                                                                              1  31.4                                                                              0.7 32.2  97.7                                                                              2.3                                                                              0.25 0.03 0.28                              125                                                                              32 31.2                                                                              2  31.7                                                                              1.5 33.2  95.5                                                                              4.5                                                                              0.25 0.06 0.31                              125                                                                              32 31.2                                                                              4  32.2                                                                              3.0 35.2  91.5                                                                              8.5                                                                              0.26 0.13 0.39                              125                                                                              32 31.2                                                                              8  33.2                                                                              6.0 39.2  84.7                                                                              15.3                                                                             0.27 0.25 0.52                              125                                                                              32 31.2                                                                              16 35.2                                                                              12.0                                                                              47.2  24.6                                                                              25.4                                                                             0.28 0.50 0.78                              125                                                                              32 0.0 32 8.0 24.0                                                                              32.0  25.0                                                                              75.0                                                                             0.06 1.00 1.06                          __________________________________________________________________________     QB* = QB × 75%                                                     

The new combination has a use rate of from 20 to 100 ppm and preferablyfrom about 25 to about 50 ppm. It could be higher; however thepossibility of sensitization would increase as the rate of useincreases.

The percentage of 651 in the 573/651 combination which shows synergy isfrom 1.2 to 25.4% with the more preferred percentage of 651 in thecombination of 573/651 being in the range of from about 2.3 to about8.5% and the most preferred percentage of 651 in the combination of573/651 being in the range of from about 2.3 to 4.5%.

These ranges of 651 are preferred (i.e. 2.3 to 8.5% and 2.3 to 4.5%)because they provide the lowest amounts of 651 and strongest synergy.

Sensitization Study

The study was conducted to determine the sensitization potential of thesynergistic combination of 3.5% 651 and 96.5% 573. The results werecompared to the sensitization potential of 886 (containing 75% 651 and25% 573) and pure 573.

These results are reported in Table 3.

The closed-patch test of Buehler Current Concepts in CutaneousToxicology, (New York: Academic Press 1970), pp. 25-40, is used toinvestigate the sensitization potential of the materials in the guineapig. The combination (1.5% total active ingredient) at aqueousconcentrations of 1000, 5000, 10000 and 15000 ppm a.i., the 886 (13.9%total active ingredient) at aqueous concentrations of 250, 500 and 750ppm a.i. and the 573 (solid containing 99.5% total active ingredient) ataqueous concentrations of 5000, 15000 and 30000 ppm a.i. were appliedtopically to the skin of guinea pigs by means of occluded patches (0.4ml per patch). All guinea pigs (10 per group at each concentration)received a total of ten 6-hr induction exposures over a 22 day period.In addition, a group of guinea pigs were treated with dry patches onlyand served as the naive control group. Two weeks after the lastinduction treatment guinea pigs induced with the combination werechallenged with 3 concentrations of the combination in distilled water(1000, 5000 and 15000 ppm a.i.), those induced with 886 were challengedwith 3 concentrations of 886 in distilled water (100, 250 and 750 ppma.i.) and those induced with 573 alone were challenged with 3concentrations of 573 in distilled water (1000, 5000 and 15000 ppma.i.). Twenty-four (24) and 48 hr after the challenge doses, theapplication sites were examined for erythema reactions and graded from 0to 3 in accordance with a pre-established scoring system. Reactionsgraded 1 or greater were judged to be skin sensitization reactions.

No erythema was observed in the naive control group following challengewith 15000 ppm a.i. of the combination, 750 ppm a.i. 886 and 15000 ppma.i. 573--the highest concentrations of each test material used forchallenge (Table 1). In guinea pigs induced with the combination atconcentrations of 1000 to 10000 ppm a.i. erythema responses wereobserved following challenge with 5000 and 15000 ppm a.i. of thecombination (2/10 to 7/10 incidence), but not with 1000 ppm a.i. Animalsinduced with 15000 ppm a.i. of the combination exhibited a minimalresponse at 1000 ppm a.i. challenge (1/10), as well as, responses at5000 and 15000 ppm a.i. challenge (4/10 and 7/10, respectively).

In guinea pigs induced with 886 biocide at 250 ppm, erythema responseswere observed at 250 and 750 ppm a.i. challenge (1/10 and 4/10,respectively), but not at 100 ppm a.i. challenge. Animals induced with886 at 500 and 750 ppm a.i. exhibited positive responses followingchallenge with 100, 250 and 750 ppm a.i. of biocide (1/10 to 6/10incidence), except with those animals induced and challenged with 750and 250 ppm a.i., respectively.

In guinea pigs induced with 573 at 5000 and 15000 ppm a.i., no erythemawas observed following challenge with 1000 ppm a.i. 573 and a minimal tolow incidence of erythema responses were observed following challengewith 5000 and 15000 ppm a.i. 573 (i.e., 1/10 to 3/10 incidence). Animalsinduced with 30000 ppm a.i. 573, exhibited a minimal to low incidence oferythema at all challenge concentrations of 573 (1/9 to 3/9).

A comparison of the sensitization potential of the synergisticcombination to that of 886 suggests that the combination is 1/20 to 1/15times as potent a sensitizer as 886. A statistical procedure was used tocompare the incidence and severity of the responses in animals induced(5000, 10000 and 15000 ppm a.i.) and challenged (15000 ppm a.i.) withthe combination to that in animals induced (250, 500 and 750 ppm a.i.)and challenged (750 ppm a.i.) with 886. The distribution of skinreaction scores in the 886 induction groups was not statisticallydifferent (p<0.20) from that in the synergistic combination inductiongroups (i.e., at 20 times higher concentrations) following challengewith the highest doses. Likewise, the sensitization potential of the 573combination was approximately 1/3 to 1/2 that of the combination (i.e.,at 5000 and 15000 ppm induction and 1000, 5000 and 15000 ppm challengefor each test material, the overall incidence was 23 out of a possible60 responses versus 7 out of a possible 60 responses for the synergisticcombination and 573, respectively).

In conclusion, the synergistic combination produced delayed contacthypersensitivity in guinea pigs, but the sensitization potential of thistest combination is 1/20 to 1/15 that of 886.

                  TABLE 3                                                         ______________________________________                                        Erythema Responses of 1 or Greater                                            at 24 and/or 48 Hours                                                         Induction Phase.sup.a                                                                        Challenge Phase.sup.b                                          ______________________________________                                                           Synergistic                                                                   Combination                                                                              886    573                                      Treatment                                                                              Dose (ppm)                                                                              15000 ppm   750 ppm                                                                             15000 ppm                                ______________________________________                                        Naive    --        0/10       0/10   0/10                                     Control.sup.c                                                                 ______________________________________                                                     Synergistic Combination                                          Treatment                                                                              Dose (ppm)                                                                               1000 ppm  5000 ppm                                                                             15000 ppm                                ______________________________________                                        Synergistic                                                                             1000     0/10       2/10   2/10                                     Combination                                                                             5000     0/10       4/10   7/10                                     Synergistic                                                                            10000     0/10       3/10   6/10                                     Combination                                                                            15000     1/10       4/10   7/10                                     ______________________________________                                                           886        886    886                                      Treatment                                                                              Dose (ppm)                                                                               100 ppm    250 ppm                                                                              750 ppm                                 ______________________________________                                        886       250      0/10       1/10   4/10                                     886       500      1/10       2/10   6/10                                     886       750      2/10       0/10   6/10                                     ______________________________________                                                           573        573    573                                      Treatment                                                                              Dose (ppm)                                                                               1000 ppm  5000 ppm                                                                             15000 ppm                                ______________________________________                                        573       5000     0/10       1/10   2/10                                     573      15000     0/10       1/10   3/10                                     573      30000     1/9        2/9    3/9                                      ______________________________________                                         .sup.a Guinea pigs received ten (6 hr.) induction exposures of the ten        materials (0.4 ml per exposure) over a 22 day period.                         .sup.b All guinea pigs received 0.4 ml of challenge doses as specified tw     weeks after the last induction dose.                                          .sup.c Guinea pigs received sham treatment only.                         

What is claimed:
 1. A microbicidal composition comprising a synergisticmixture the first component of which is in the range of from about 1.2to 25.4% of 5-chloro-2-methyl-4-isothiazolin-3-one and the secondcomponent of which is in the range of from about 74.6 to about 98.8% of2-methyl-4-isothiazolin-3-one.
 2. The microbicidal composition of claim1 wherein the 5-chloro-2-methyl-4-isothiazolin-3-one is in the range offrom about 2.3 to 8.5% and the 2-methyl-4-isothiazolin-3-one is in therange of from about 91.5 to about 97.7%.
 3. The microbicidal compositionof claim 2 wherein the 5-chloro-2-methyl-4-isothiazolin-3-one is in therange of from about 2.3 to about 4.5% and the2-methyl-4-isothiazolin3-one is in the range of from about 95.5 to about97.7%.